U.S. patent application number 09/815346 was filed with the patent office on 2002-02-14 for lipopeptide adjuvants.
Invention is credited to Agrawal, Babita, Longenecker, Michael B., Parker, Joanne.
Application Number | 20020018806 09/815346 |
Document ID | / |
Family ID | 22706736 |
Filed Date | 2002-02-14 |
United States Patent
Application |
20020018806 |
Kind Code |
A1 |
Agrawal, Babita ; et
al. |
February 14, 2002 |
Lipopeptide adjuvants
Abstract
Vaccine compositions containing a MUC-1-based adjuvant and an
antigen are useful in treating and preventing disorders such as
cancer and viral diseases. Exemplary compositions contain a
25-amino acid lipopeptide adjuvant and an antigen of interest in
association with a liposome.
Inventors: |
Agrawal, Babita; (Edmonton,
CA) ; Longenecker, Michael B.; (Edmonton, CA)
; Parker, Joanne; (Edmonton, CA) |
Correspondence
Address: |
Bernhard D. Saxe
FOLEY & LARDNER
Washington Harbour
3000 K Street, N.W., Suite 500
Washington
DC
20007-5109
US
|
Family ID: |
22706736 |
Appl. No.: |
09/815346 |
Filed: |
March 23, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60191736 |
Mar 24, 2000 |
|
|
|
Current U.S.
Class: |
424/450 ;
424/204.1; 424/234.1; 424/277.1 |
Current CPC
Class: |
A61K 2039/55516
20130101; A61K 2039/55555 20130101; A61K 39/39 20130101; A61P 37/00
20180101; A61P 37/04 20180101 |
Class at
Publication: |
424/450 ;
424/204.1; 424/277.1; 424/234.1 |
International
Class: |
A61K 009/127; A61K
039/12; A61K 039/02; A61K 039/00 |
Claims
What is claimed is:
1. A vaccine composition, comprising a MUC-1-based adjuvant peptide
and an antigen.
2. A vaccine according to claim 1, wherein said adjuvant is from
about 12 to about 25 amino acids long.
3. A vaccine according to claim 1, wherein said adjuvant is from
about 9 to about 11 amino acids long.
4. A vaccine according to claim 2, wherein said adjuvant is
lipid-modified.
5. A vaccine according to claim 2, wherein the adjuvant is BPI-217
or a derivative thereof.
6. A vaccine according to claim 2, wherein the adjuvant is BPI-228
or a derivative thereof.
7. A vaccine according to claim 2, wherein the adjuvant is BPI-132
or a derivative thereof.
8. A vaccine according to claim 2, wherein the adjuvant is BPI-148
or a derivative thereof.
9. A vaccine according to claim 2, wherein the adjuvant is BPI-216
or a derivative thereof.
10. A vaccine according to claim 1, wherein said antigen is
selected from the group consisting of viral antigens, tumor
antigens, parasite antigens and bacterial antigens.
11. A vaccine according to claim 1, wherein said antigen is
lipid-modified.
12. A vaccine according to claim 11, wherein said antigen is a
selected from the group consisting of viral antigens, tumor
antigens, parasite antigens and bacterial antigens.
13. A vaccine according to claim 1, further comprising a delivery
vehicle.
14. A vaccine according to claim 13, wherein said delivery vehicle
is a liposome.
15. A vaccine according to claim 1, wherein said adjuvant and said
antigen are covalently linked to one another.
16. A method of stimulating the immune response of a patient,
comprising administering to said patient the vaccine of claim
1.
17. A method of stimulating the immune system of a patient,
comprising contacting ex vivo a T-cell from the patient with the
vaccine of claim 1 and administering to the patient the contacted
cells.
Description
BACKGROUND OF THE INVENTION
[0001] Immunotherapy or vaccine therapy approach is an attractive
form of therapy for certain viral, bacterial infections and various
cancers. However, immunotherapy for these diseases is restricted
partially due to the fact that a number of target antigens
(peptides, glycopeptides, lipids, lipopeptides, carbohydrates etc.)
are poorly immunogenic or induce non-desirable type of immune
responses, e.g., antibody response only or type 2 T cell responses
only. This specific skew in immune response towards a specific
antigen is in part dependent upon the major histocompatibility
complex molecules, in vivo environment, pre-exposure to another
infection and T cell repertoire etc.
[0002] An ideal vaccine antigen should contain both B and T cell
epitopes. An effective immune response would consist of both
antibody and cytotoxic T cell mediated effector functions.
Generation of both antibody and cytotoxic T cell responses against
a given antigen requires that a strong T helper cell response is
generated. T helper cell responses are provided by CD4+ T cells
that recognize fragments of peptide antigens in context of MHC
class II molecules on the surface of antigen presenting cells
(APCs). Most of the processed forms of peptide antigens are only
able to be presented by one or a few alleles of MHC haplotypes.
Therefore, T helper response to a given antigenic peptide becomes
strictly under control of genetic makeup of an individual.
Therefore, inclusion of a helper epitope in most cases would become
restricted to one or a few restricted haplotypes of MHC out of a
divergent population with highly polymorphic MHC molecules. This
genetically restricted T helper cell stimulatory activity of
peptide antigens presents a serious obstacle and consequently such
T helper epitopes become of limited practical value as a vaccine
candidate for majority of an outbred population.
[0003] In order to avoid the above limitation with T helper peptide
epitopes, large proteins have been utilized as carrier molecules.
However, use of large proteins as carriers is expensive, variable
and may result in adverse effects upon repeated
administrations.
[0004] Therefore, identification of T helper epitope peptides that
can be presented in context of a vast majority of haplotypes of MHC
class II molecules and therefore induce strong CD4+ T helper
responses in majority of outbred human population, is highly
desirable. Such T helper peptide epitopes are generally referred to
as "Promiscuous" or "Permissive"T helper epitopes. Such promiscuous
T helper epitopes have been defined and identified before, e.g.,
tetanus toxoid peptide, Plasmodium falciparum (pfg27), Lactate
dehydrogenase, HlVgp120 etc. (Infect. Immun, 1998; 66:3579-3590, C
E Contreas et al; J. A.I.D.S. Human Retrovirol 1997; 14:91-101, P.
Gaudebout et al; J. Mol. Recog. 1993; 6:81-94, P T Kaumaya et al;
J. Immunol.1992;148:907-913, J. Fern and M F Good).
[0005] Some of these promiscuous T helper epitopes have also been
shown in conjunction with other antigens to induce strong B cells
response to a given antigen as well as to bypass certain haplotype
restricted immune responses (J. Mol. Recog., 1993, 6:81-94, P T
Kaumaya et al).
[0006] A need exists in the art, therefore, for promiscuous
epitopes useful in enhancing and generalizing the immune response
against otherwise inferior antigens.
SUMMARY OF THE INVENTION
[0007] It is an object of the invention to provide compositions and
methods that overcome the deficiencies of the art.
[0008] According to this object, the invention provides a vaccine
composition, containing a MUC-1-based adjuvant peptide and an
antigen. In one aspect, the adjuvant is from about 12 to about 25
amino acids long, yet in other it is from about 9 to about 11 amino
acids long. The adjuvant may be lipid or carbohydrate modified. In
addition, the adjuvant and antigen may be covalently linked or part
of a fusion protein. Possible antigens, which also may be
lipid-modified, include viral antigens, tumor antigens, parasite
antigens and bacterial antigens. In a preferred aspect, the vaccine
contains a liposome.
[0009] Also according to this object, the invention provides a
method of stimulating the immune response of a patient. In one
embodiment, the method involves administering to a patient an
inventive vaccine. In an alternative embodiment, the method entails
contacting ex vivo a T-cell and/or and APC from a patient with an
inventive vaccine and administering T-cell and/or an APC to the
patient.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0010] We have identified a promiscuous T helper epitope from the
peptide sequence of extracellular tandem repeat domain of MUC1
mucin. This promiscuous T helper epitope could be used
therapeutically in conjunction with other poorly immunogenic or
non-immunogenic antigens to induce strong immune responses. This
epitope could also be used to bypass MHC haplotype restriction for
certain antigens.
[0011] Accordingly, the invention relates to vaccine compositions
and their use in stimulating a patient's immune system. The present
vaccines have two basic components: a promiscuous MUC-1-derived
T-cell antigen (and "adjuvant" for the purposes of the invention)
and a non-MUC-1-antigen. The promiscuous MIUC-1 -derived antigen
acts as an adjuvant to generate or enhance an immune response to
the antigen upon administration to a patient.
[0012] Because the inventive vaccine compositions incorporate a
"promiscuous" or "permissive" T-cell antigen derived from MUC-1,
they are particularly effective at generating an immune response to
an antigen against which the patient otherwise would not respond or
would not respond to therapeutically or prophylactically effective
levels.
[0013] As used herein with reference to MUC-1 -derived peptides,
"promiscuous" and "permissive" are used interchangeably to indicate
a general lack of specificity for any particular HLA molecule. Such
a peptide may bind to class I or class II molecules and among the
different subclasses of class I and class II molecules. The skilled
artisan will be familiar with assays for measuring promiscuity.
These promiscuous MUC-1-derived peptides are also referred to
herein as "adjuvants."
[0014] The promiscuous MUC-1-derived peptides useful in the present
invention are used in conjunction with a target antigen molecule,
which is a non-MUC-1-antigen. This target antigen can be from any
source against which immunity is sought. Due to their general
stimulatory character, the promiscuous MUC-1-derived peptides are
useful adjuvants in generating or enhancing an immune response
against the target antigen.
[0015] Promiscuous MUC-1-Derived Peptides (Adjuvants)
[0016] The promiscuous MUC-1 -derived peptides (adjuvants) are
based on the following amino acid sequence:
STAPPAHGVTSAPDTRAPGSTAPP. This core region may also be modified to
generate "derivatives," as described in detail below, in ways which
the derivative retains the promiscuous nature of the molecule. For
example, it may be shorted from the C-terminus to about 12 amino
acids and promiscuity should be retained. The basic sequence also
may be shorted to about 9 amino acids from the C-terminus and
promiscuity among class I molecules should be retained, however,
such molecules are expected to lose class II binding capability.
Thus, derivatives from about 12 to about 24 amino acids are
preferred, because they stimulate both class I and class II
molecules, with about 15 to about 20 amino acids providing a quite
suitable range. On the other hand, where only class I-associated
immunostimulation is desired (e.g., CTL function), it may be
desirable to utilize adjuvant molecules having from about 9 to
about 11 amino acids. In addition, the following adjuvant
"derivatives" are contemplated.
[0017] The basic sequence above represents slightly more than a
single direct repeat (of up to about a hundred) from the native
MUC-1 molecule. Thus, while the sequence is presented as beginning
with STAPP, and such molecules are preferred, the invention also
contemplates other permutations, beginning at other amino acids,
but falling within the size parameters outlined herein. For
example, with reference to the above core sequence, molecules could
begin TAPPA, APPAH, PPAHG, and so on.
[0018] Moreover, one or more amino acids of the core sequence may
be altered, preferably in a conservative manner known in the art,
such that the requisite promiscuity is maintained, or even
enhanced. Typical substitutions may be made among the following
groups of amino acids: (a) G, A, V, L and I; (b) G and P; (c) S, C,
T, M; (d) F, Y, and W; (e) H, K and R; and (f) D, E, N, and Q. Some
preferred substitutions may be made among the following groups: (i)
S and T; (ii) P and G; and (iii) A, V, L and I.
[0019] Preferred adjuvants are modified with at least one lipid
molecule. Exemplary lipid moieties include, but are not limited to,
palmitoyl, myristoyl, stearoyl and decanoyl groups or, more
generally, any C.sub.2 to C.sub.30 saturated, monounsaturated or
polyunsaturated fatty acyl group. The serine residues within the
MUC I core sequence offer convenient sites where lipid molecules
can be attached. An example of such an adjuvant is (1) BP1-217 with
two myristyl lipids attached to two serines at the carboxy terminus
of the core peptidic sequence; (2) BP1-228 with only one myristyl
lipid attached to a carboxy terminal serine;or MUC I peptide, (3)
BP1-132 with two palmitate lipid molecules attached to two adjacent
carboxy terminal lysine amino acid residue; or (4) BPI-148 with one
palmitate lipid molecule attached to a carboxy terminal lysine
amino acid residue.
[0020] BP1-217: GVTSAPDTRPAPGSTAS(myristyl)S(myristyl)L
[0021] BP1-228: GVTSAPDTRPAPGSTAS(myristyl)L
[0022] BP1-132:
TAPPAHGVTSAPDTRPAPGSTAPPK(palmitate)K(palmitate)G
[0023] BPI-148 STAPPAHGVTSAPDTRPAPGSTAPP-Lys(Palmitate)
[0024] Adjuvants also may be glycosylated, partially glycosylated,
or attached to a carbohydrate according to methods known in the art
or modified with large molecular weight polymers, such as
polyethylene glycols. An example of such an adjuvant is BPI-216
glycolipopeptide. BPI-216 has two myristyl lipids attached to two
serines at the carboxy terminus of the peptide sequence and a Tn
carbohydrate O-linked to threonine and serine of the peptide at the
GVTS sequence of the MUC1 tandem repeat. Tn carbohydrate antigen is
found on a variety epithelial cells derived form adenocarcinomas of
the breast, colon, pancreas. It is also associated with Tcell
Lymphomas.
[0025] BP1-216 GVT(Tn)S(Tn)APDTRPAPGSTAS(Myristyl)S(Myristyl)L
[0026] For convenience in making chemical modifications, it is
sometimes useful to include in a MUC-1 peptide one or more amino
acids having a side chain amenable to modification. A preferred
amino acid is lysine, which may readily be modified at the
.epsilon.-amino group. Side chain carboxyls of aspartate and
glutamate are readily modified, as are serine, threonine and
tyrosine hydroxyl groups, the cystine sulfhydryl group and the
histidine amino group. Such additional amino acids are not included
within the size parameters provided above. Thus, while MUC-1
derived peptides may be, for example, from about 12 to about 24
amino acids, the addition of a lysine would alter the size range
from about 13 to about 25 amino acids. Likewise, the addition to
two modifiable amino acids to the molecules ranging from about 15
to about 20 amino acids would give a range of from about 17 to
about 22 amino acids, and so on.
[0027] Antigens
[0028] The present vaccines apply generally to a great variety of
antigens, which may be of nearly any chemical constitution.
Exemplary antigens can be derived from peptides, carbohydrates,
lipids and especially combinations thereof. Particularly important
antigens are peptides, lipopeptides and glycopeptides. Idiotypic
and antiidiotypic antigens are specifically included. MUC-1
antigens are not included in the present usage of the term.
Lipid-modified peptide antigens (lipopeptide antigens) are a
preferred type of antigen.
[0029] Antigens against which it would be highly advantageous to
use the subject vaccines include tumor antigens. Tumor antigens are
usually native or foreign antigens which are correlated with the
presence of a tumor. Inasmuch as tumor antigens are useful in
differentiating abnormal from normal tissue, they are useful as a
target for therapeutic intervention.
[0030] Tumor antigens are well known in the art. Indeed, several
examples are well-characterized and are currently the focus of
great interest in the generation of tumor-specific therapies.
Non-limiting examples of tumor antigens are carcinoembryonic
antigen (CEA), prostate specific antigen (PSA), melanoma antigens
(MAGE, BAGE, GAGE), and mucins, such as MUC-1.
[0031] In another embodiment, the antigen is a parasite-associated
antigen, such as an antigen associated with leishmania, malaria,
trypanosomiasis, babesiosis, or schistosomiasis. Suitable
parasite-associated epitopes include, but are not limited to, the
following.
1 Parasite Epitope References Plasmodium Falciparum (NANP)3 Good et
al. (1986) (Malaria) J. Exp. Med. 164:655 Circumsporoz. Good et al.
(1987) Protein Science 235:1059 AA 326-343 Leishmania donovani
Repetitive peptide Liew et al. (1990) J. Exp. Med. 172:1359
Leishmani major EAEEAARLQA (code) Toxoplasma gondii P30 surface
protein Darcy et al. (1992) J. Immunolog. 149:3636 Schistosoma
mansoni Sm-28GST antigen Wolowxzuk et al. (1991) J. Immunol
146:1987
[0032] In another embodiment, the epitope is a viral epitope, such
as an epitope associated with human immunodeficiency virus (HIV),
Epstein-Barr virus (EBV), or hepatitis. Suitable viral epitopes
include, but are not limited to:
2 Virus Epitope Reference HIV gp120 V3 loop, 308-331 Jatsushita, S.
et al. (1988) J. Viro. 62:2107 HIV GP120 AA 428-443 Ratner et al.
(1985) Nature 313:277 HIV gp120 AA 112-124 Berzofsky et al. (1988)
Nature 334:706 HIV Reverse transcriptase Hosmalin et al. (1990)
PNAS USA 87:2344 Flu nucleoprotein Townsend et at. (1986) AA
335-349, 366-379 Cell 44:959 Flu haemagglutinin Mills et al. (1986)
AA48-66 J. Exp. Med. 163:1477 Flu AA111-120 Hackett et al. (1983)
J. Exp. Med 158:294 Flu AA114-131 Lamb, J. and Green N. (1983)
Immunology 50:659 Epstein-Barr LMP43-53 Thorley-Lawson et al.
(1987) PNAS USA 84:5384 Hepatitis B Surface Ag Milich et al. (1985)
AA95-109; J. Immunol. 134:4203 AA 140-154 Pre-S antigen Milich, et
al. (1986) AA 120-132 J. Exp. Med. 164:532 Herpes simplex gD
protein Jayaraman et al. (1993) AA5-23 J. Immunol. 151:5777 gD
protein Wyckoff et al. (1988) AA241-260 Immunobiology 177:134
Rabies glycoprotein MacFarlan et al. (1984) AA32-44 J. Immunol.
133:2748
[0033] The epitope may also be associated with a bacterial antigen.
Suitable epitopes include, but are not limited to:
3 Bacteria Epitope ID Reference Tuberculosis 65Kd protein Lamb et
al. (1987) AA112-126 EMBO J. 6:1245 AA163-184 AA227-243 AA242-266
AA437-459 Staphylococcus nuclease protein Finnegan et al. (1986)
AA61-80 J. Exp. Med. 164:897 E. coli heat stable enterotoxin
Cardenas et al. (1993) Infect. Immunity 61:4629 heat liable
enterotoxin Clements et al. (1986) Infect. Immunity 53:685 Shigella
sonnei form I antigen Formal et al. (1981) Infect. Immunity
34:746
[0034] Vaccine Compositions of the Invention
[0035] The inventive compositions may be formulated for
administration in a variety of ways. The pharmaceutical
compositions of the invention generally contain an immunologically
effective amount of an adjuvant and an antigen. Preferably, the
adjuvant and antigen are admixed with a pharmaceutically effective
vehicle (excipient). In one embodiment, the adjuvant and the
antigen are covalently linked to one another. Such linking may be
accomplished using methods known to the skilled worker (e.g.,
production as a fusion protein or linking using chemical
linkers).
[0036] Guidance in preparing suitable formulations and
pharmaceutically effective vehicles, can be found, for example, in
REMINGTON'S PHARMACEUTICAL SCIENCES, chapters 83-92, pages
1519-1714 (Mack Publishing Company 1990) (Remington's), which are
hereby incorporated by reference.
[0037] Preferred vehicles include liposomes. When liposomes are
used, conventional vaccine components like Freund's adjuvant,
Keyhole Limpet Haemocyanin ("KLH"), Lipid A, monophosphoryl Lipid A
("MPLA"), and the like are optional; the invention specifically
contemplates indpendently their presence or absense. For general
details on liposomes, see, for example, Remington's at 1691-92.
Techniques for preparation of liposomes and the formulation (e.g.,
encapsulation) of various molecules, including peptides and
oligonucleotides, with liposomes are well known to the skilled
artisan. Liposomes are microscopic vesicles that consist of one or
more lipid bilayers surrounding aqueous compartments. See,
generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.
Infect. Dis. 12 (Suppl. 1): S61 (1993) and Kim, Drugs 46: 618
(1993). Liposomes are similar in composition to cellular membranes
and as a result, liposomes generally can be administered safely and
are biodegradable.
[0038] Depending on the method of preparation, liposomes may be
unilamellar or multilamellar, and can vary in size with diameters
ranging from 0.02 .mu.m to greater than 10 .mu.m. A variety of
agents can be encapsulated in liposomes. Hydrophobic agents
partition in the bilayers and hydrophilic agents partition within
the inner aqueous space(s). See, for example, Machy et al.,
LIPOSOMES IN CELL BIOLOGY AND PHARMACOLOGY (John Libbey 1987), and
Ostro et al., American J. Hosp. Pharm. 46: 1576 (1989).
[0039] Liposomes can adsorb to virtually any type of cell and then
release the encapsulated agent. Alternatively, the liposome fuses
with the target cell, whereby the contents of the liposome empty
into the target cell. Alternatively, an absorbed liposome may be
endocytosed by cells that are phagocytic. Endocytosis is followed
by intralysosomal degradation of liposomal lipids and release of
the encapsulated agents. Scherphof et al., Ann. N.Y. Acad. Sci.
446: 368 (1985). Irrespective of the mechanism or delivery,
however, the result is the intracellular disposition of the
associated therapeutic.
[0040] Anionic liposomal vectors have also been examined. These
include pH sensitive liposomes which disrupt or fuse with the
endosomal membrane following endocytosis and endosome
acidification.
[0041] Among liposome vectors, cationic liposomes are the most
studied, due to their effectiveness in mediating mammalian cell
transfection in vitro. They are often used for delivery of nucleic
acids, but can be used for delivery of other therapeutics, be they
drugs or hormones.
[0042] Liposomes are preferentially phagocytosed into the
reticuloendothelial system. However, the reticuloendothelial system
can be circumvented by several methods including saturation with
large doses of liposome particles, or selective macrophage
inactivation by pharmacological means. Classen et al., Biochim.
Biophys. Acta 802: 428 (1984). In addition, incorporation of
glycolipid- or polyethylene glycol-derivatised phospholipids into
liposome membranes has been shown to result in a significantly
reduced uptake by the reticuloendothelial system. Allen et al.,
Biochim. Biophys. Acta 1068: 133 (1991); Allen et al., Biochim.
Biophys. Acta 1150: 9 (1993).
[0043] Cationic liposome preparations can be made by conventional
methodologies. See, for example, Feigner et al, Proc. Nat'l Acad.
Sci USA 84:7413 (1987); Schreier, J. of Liposome Res. 2:145 (1992);
Chang et al. (1988), supra. Commercial preparations, such as
Lipofectin (Life Technologies, Inc., Gaithersburg, Md. USA), also
are available. The amount of liposomes and the amount of DNA can be
optimized for each cell type based on a dose response curve.
Feigner et al., supra. For some recent reviews on methods employed
see Wassef et al., Immunomethods 4: 217-222 (1994) and Weiner, A.
L., Immunomethods 4: 217-222 (1994).
[0044] Other suitable liposomes that are used in the methods of the
invention include multilamellar vesicles (MLV), oligolamellar
vesicles (OLV), unilamellar vesicles (UV), small unilamellar
vesicles (SUV), medium-sized unilamellar vesicles (MUV), large
unilamellar vesicles (LUV), giant unilamellar vesicles (GUV),
multivesicular vesicles (MVV), single or oligolamellar vesicles
made by reverse-phase evaporation method (REV), multilamellar
vesicles made by the reverse-phase evaporation method (MLV-REV),
stable plurilamellar vesicles (SPLV), frozen and thawed MLV
(FATMLV), vesicles prepared by extrusion methods (VET), vesicles
prepared by French press (FPV), vesicles prepared by fusion (FUV),
dehydration-rehydration vesicles (DRV), and bubblesomes (BSV). The
skilled artisan will recognize that the techniques for preparing
these liposomes are well known in the art. See COLLOIDAL DRUG
DELIVERY SYSTEMS, vol. 66 (J. Kreuter, ed., Marcel Dekker, Inc.
1994).
[0045] An example of a liposomal vaccine is BLP25. BLP25 is
comprised of a liposomal delivery system, an antigen, and the
BPI-148 lipopeptide adjuvant.
[0046] Other forms of delivery particle, for example, microspheres
and the like, also are contemplated.
[0047] Therapeutic and Prophylactic Methods of the Invention
[0048] The methods of the invention may be accomplished in vivo or
ex vivo. In vivo approaches generally entail administering to a
patient an immunogenically effective amount of an inventive vaccine
composition. An effective amount is an amount sufficient to enhance
a weak immune response to the antigen or an amount sufficient to
generate an immune response where, absent the adjuvant, a response
could not be generated.
[0049] The inventive methods are useful in both therapeutic and
prophylatic contexts. Thus, if a patient is suffering from a
disorder, the methods may be used to mitigate that suffering.
Likewise, used prophylactically (prior to disease onset), the
present methods can be used to prevent or lessen the severity of a
disorder.
[0050] In an ex vivo approach, the inventive vaccines may be used
to generate an immune response ex vivo. In particular, immune cells
(peripheral blood lymphocytes or isolated dendritic cells, for
example) from a patient may be used to prime a patient's T-cells in
vitro. In general, antigen presenting cells are loaded with an
inventive vaccine composition and the resultant loaded cells are
used as antigen presenting cells to generate antigen-specific
T-cells, which may then be infused back into a patient in need of
treatment. The artisan will be familiar, from the literature, with
approaches such as this. The present vaccine compositions can be
used in any such method.
[0051] The following examples are for illustrative purposes and are
not meant to be limiting.
EXAMPLES
Example 1
T Cell Response to BLP25 in Normal Donors
[0052] This example demonstrates that BLP25 generates a
surprisingly strong immune response, which is suggestive of the
promiscuous nature of the antigen. Buffy coats were collected from
Canadian Blood Servies from normal donors. Buffy coats were used to
purify monocytes (Miltenyi MACS column for CD14+ cells) and T cells
(nylon wool columns). The CD14+ monocytes were cultured in presence
of GM-CSF (50 ng/ml) and IL-4 (10 ng/ml) for 3 days. At this time,
the immature dendritic cells were (DCs) were harvested and further
cultured for additional 3 days in presence of media, liposomes
containing BLP25 at 400 .mu.g/ml or no antigen and Avanti lipid A.
After this culture, the antigen loaded DCs were washed, irradiated
and added to autologous T cells for 5-6 days of culture in 96 well
flat bottom plates. At this time, the wells were pulsed with
3H-thymidine overnight and 3H-Tdr incorporation into proliferating
T cells was determined by counting in a liquid scintillation
counter. FIG. 1 represents one experiment out of 6 reproduced
experiments (all from different donors). In all of these 6 donors,
strong T cell proliferative response was observed suggesting
promiscuous nature of BLP25.
Example 2
T Cell Proliferative Response of Non Small Cell Lung Cancer (NSCLC)
Patients Against BLP25
[0053] In a phase II clinical trial, eight NSCLC patients were
immunized with liposomal BLP25 vaccine at 1000 ug/injection on a
weekly basis for eight weeks. Blood was drawn a week after every
two injections and peripheral blood mononuclear cells were
separated by FicoII method. Proliferative responses were determined
in response to soluble BLP25 in vitro cultures. As indicated in
Table I, PBMCs from six out of eight immunized patients showed a
strong proliferative response against BLP25. These results further
confirm promiscuous T helper nature of BLP25.
Example 3
Ascertaining Antigen Promiscuity
[0054] In order to determine the adjuvant activity of BLP25, a
liposome containing BLP25, a 9 mer telomerase peptide or a
glycopeptide antigen are formulated and used to stimulate human T
cells in vitro using dendritic cells as efficient antigen
presenting cells (APCs). T cell responses are determined against
both BLP25 and the telomerase peptide cytotoxic activity as a
measure of immune response. An enhancement of the response against
telomerase in the presence of BLP25 is indicative of the adjuvant
effect.
[0055] In general, PCT/US98/09288; Agrawal et al., Int'l
Immunol.10:1907-16 (1998); and Agrawal et al., Cancer Res.
55:5151-56 (1 998) provide suitable methods, and those disclosures
are hereby incorporated by reference, in their entirety.
[0056] Peotides
[0057] Telomerase-derived antigenic peptides used in this
experiment: RLVDDFLLV, ELLRSFFYV and ILAKFLHWL.
[0058] Preparation of Liposomes
[0059] The bulk liquid composition of liposomes consist of
dipalmitoyl phosphatidyl choline (DPPC), cholesterol (Chol) and
dimyristoyl phosphatidyl glycerol (DMPG) in a molar ratio of
3:1:0.25 and contain Lipid A at a concentration of 1% (w/w) of bulk
lipid. Synthetic telomerase peptides are present in the aqueous
phase during liposome formation at a concentration of 0.7 mg/ml
BLP25 also is present, except for a control sample. The formulated
product contains 2 mg of bulk lipid, 20 .mu.g Lipid A, with or
without about 40 .mu.g BLP25, and about 20 .mu.g of peptide per 100
.mu.l.
[0060] General Procedures for Loading APCs with
Liposome-encapsulated Peptide
[0061] Briefly, to 2-10.times.10.sup.6 human dendritic cells in 0.9
mL AIM-V media, one dose of liposome containing peptide formulation
is added and the cells were incubated overnight at 37.degree. C.
with CO.sub.2 supplemented incubator. After incubation, the cells
are treated with mitomycin C or .gamma.-irradiated (3000 rads)
followed by washing with AIM-V media.
[0062] Cytotoxic T Lymphocyte Assays
[0063] For the CTL assay, T-cells are grown for five weeks in bulk
cultures. At the end of two weeks, live T-cells are harvested from
flasks and counted. The targets are mutant T2 cells. Houbiers et
al., Eur. J. Immunol 23:2072-2077 (1993); Stauss et al., Proc.
Natl. Acad. Sci. U.S.A. 89:7871-7875 (1992). T2 cells are loaded
overnight at 37.degree. C. in 7% CO.sub.2, with or without BLP25,
with various the telomerase synthetic peptides at 200 .mu.M in
presence of 8 .mu.g exogenous .beta.2 microglobulin. Houbiers et
al., supra; Stauss et al., supra. The peptide-loaded T2 target
cells are loaded with .sup.51Cr (using NaCrO.sub.4) for 90 minutes
and washed. CTL assays are performed as previously described.
Agrawal et al., J. Immunol. 156:2089 (1996). Percent specific
killing is calculated as: experimental release-spontaneous
release/maximum release-spontaneous release.times.100. The effector
versus target ratios used is 50:1, 25:1, 10:1 and 5:1. Each group
is set up in four replicate and mean percent specific killing is
calculated.
Example 4
Demonstration of T-cell Promiscuity of BPI-148 in Unimmunized
Humans
[0064] This example demonstrates that BPI-148 generates a strong
immune response, which is suggestive of the promiscuous nature of
BPI-148. Ficoll-Paque (Pharmacia; Uppsala, Sweden) separated
peripheral blood monocyte cells were isolated from the peripheral
circulatory system and cultured in AIM V (life Technologies,
Gaithersberg, Md.) plus 5% human AB serum at 3.times.10.sup.5/well
in 4-5 replicates in the presence or absence of BPI-148 or tetanus
toxoid lipopeptide for 5-6 days in 96 well flat-bottom plates. At
this time, the wells were pulsed with 1 .mu.Ci/well
.sup.3H-thymidine (Amersham Canada Limited; Oakville, Ontaria) for
18 hours and .sup.3H-Tdr incorporation into DNA was measured after
harvesting the cells onto filter and counting in liquid
scintillation counter. The results for this experiment are shown
below in Table 2 A strong T cell proliferative response was
observed suggesting the promiscuous nature of BPI-148.
4 TABLE 2 Lipopeptide in culture *Responder/Total BPI-148 10/22
Tetanus toxoid 10/21
[0065] Responders are defined as peripheral blood mononuclear cells
giving .gtoreq.2 S.I. (S.I.=counts per minute in the presence of
antigen/counts per minute in the absence of antigen, media
only).
* * * * *